*Target (protein/gene name): Inosine 5 Monophosphate dehygrogenase *NCBI Gene # or RefSeq#: GuaB, 1083772 *Protein ID (NP or XP #) or Wolbachia#: GI: 349587863 *Organism (including strain): Bacillus anthracis str.Ames Etiologic Risk Group (see link below): Category A pathogen, US Dept of Health */Disease Information (sort of like the Intro to your Mini Research Write up): Anthrax derives from the gram-positive bacterium Bacillus anthracis with symptoms ranging from a rash to acute respiratory syndrome depending on the method of contraction. Bacillus anthracis is a bacterium which is capable of bioterrorist weaponization and is listed as a Category A pathogen by the US government. General public vaccination against B.anthracis is unrealistic given associated expenses, and current medications for the most deadly form of contraction--inhalation-- are medically intensive, requiring IV therapy and the assemblage of massive drug stockpiles. Given B.anthracis’ capacity for aerosol weaponization, the FDA has announced the need to create a prophylactic drug capable of being mass administered to individuals who are thought to have been exposed. By targeting B.anthracis Ionosine-5-monophosphate dehydrogenase (BaI5MD), an enzyme that is essential in B.anthracis DNA and RNA synthesis, our research aims to virtually screen large databases of drug-like molecules in order to identify potential inhibitors of BaI5MD. After cloning the BaI5MD gene and purifying BaI5MD protein, molecules which are predicted to bind with high affinity to BaI5MD can be selectively tested in vitro using spectrophotometric UV enzyme assays. Successful identification of novel BaI5MD inhibitors will then allow in-vivo testing of inhibition, followed by human cell toxicity tests. These developments would introduce the potential formulation of pharmaceutical therapies specifically targeted against B.anthracis, ideally eliminating it as a bioterrorist threat.
Link to TDR Targets page (if present): N/A Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) Essentiality of this protein: Essential Reference: Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Complex of proteins?: Tetramer Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.8 in T.cruzi, 1 in P.falciparum *EC#: 1.1.1.205 Link to BRENDA EC# page: Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 3 mM EDTA, and 1 mM DTT; Reactions were initiated by the addition of IMPDH to a final concentration of 50 nM. The production of NADH was measured by monitoring the increase in absorbance at 340 nm (ε = 6.22 mM−1 cm−1) using a Shimadzu BioSpec-1601, Hitachi U-2000, or Cary 100 Bio spectrophotometer. -- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3836674/#R7 -- links to assay reagents (substrates) pages. --- List cost and quantity of substrate reagents, supplier, and catalog # Ligand(s) determined from virtual screening. NADH
catalog #: N4505
supplier: Sigma-Aldrich
Cost: $67.30 for 100MG Structure (PDB or Homology model) -- PDB #: 3TSB
3USB - docked with IMP
Current Inhibitors: mycophenolic acid, tiazofurin, ribavirin , mizoribine, rapamycin
Expression Information (has it been expressed in bacterial cells): yas...
Purification Method: Ni-NTA (has 6X His Tag) Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): mhhhhhhssgvdlgtenlyfqsnamweskfvkegltfddvllvpaksdvlprevsvktvl seslqlniplisagmdtvteadmaiamarqgglgiihknmsieqqaeqvdkvkrsesgvi sdpffltpehqvydaehlmgkyrisgvpvvnnlderklvgiitnrdmrfiqdysikisdv mtkeqlitapvgttlseaekilqkykieklplvdnngvlqglitikdiekviefpnsakd kqgrllvgaavgvtadamtridalvkasvdaivldtahghsqgvidkvkevrakypslni iagnvataeatkalieaganvvkvgigpgsicttrvvagvgvpqltavydcatearkhgi pviadggikysgdmvkalaagahvvmlgsmfagvaespgeteiyqgrqfkvyrgmgsvga mekgskdryfqegnkklvpegiegrvpykgpladtvhqlvgglragmgycgaqdleflre naqfirmsgaglleshphhvqitkeapnysl *length of your protein in Amino Acids: 511aa Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 55kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 26485 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): link to DNA Works output text file
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: GuaB, 1083772
*Protein ID (NP or XP #) or Wolbachia#: GI: 349587863
*Organism (including strain): Bacillus anthracis str.Ames
Etiologic Risk Group (see link below): Category A pathogen, US Dept of Health
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Anthrax derives from the gram-positive bacterium Bacillus anthracis with symptoms ranging from a rash to acute respiratory syndrome depending on the method of contraction. Bacillus anthracis is a bacterium which is capable of bioterrorist weaponization and is listed as a Category A pathogen by the US government. General public vaccination against B.anthracis is unrealistic given associated expenses, and current medications for the most deadly form of contraction--inhalation-- are medically intensive, requiring IV therapy and the assemblage of massive drug stockpiles. Given B.anthracis’ capacity for aerosol weaponization, the FDA has announced the need to create a prophylactic drug capable of being mass administered to individuals who are thought to have been exposed. By targeting B.anthracis Ionosine-5-monophosphate dehydrogenase (BaI5MD), an enzyme that is essential in B.anthracis DNA and RNA synthesis, our research aims to virtually screen large databases of drug-like molecules in order to identify potential inhibitors of BaI5MD. After cloning the BaI5MD gene and purifying BaI5MD protein, molecules which are predicted to bind with high affinity to BaI5MD can be selectively tested in vitro using spectrophotometric UV enzyme assays. Successful identification of novel BaI5MD inhibitors will then allow in-vivo testing of inhibition, followed by human cell toxicity tests. These developments would introduce the potential formulation of pharmaceutical therapies specifically targeted against B.anthracis, ideally eliminating it as a bioterrorist threat.
Link to TDR Targets page (if present): N/A
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
Essentiality of this protein: Essential Reference:
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
Complex of proteins?: Tetramer
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
0.8 in T.cruzi, 1 in P.falciparum
*EC#: 1.1.1.205
Link to BRENDA EC# page:
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
50 mM Tris-HCl (pH 8.0), 100 mM KCl, 3 mM EDTA, and 1 mM DTT; Reactions were initiated by the addition of IMPDH to a final concentration of 50 nM. The production of NADH was measured by monitoring the increase in absorbance at 340 nm (ε = 6.22 mM−1 cm−1) using a Shimadzu BioSpec-1601, Hitachi U-2000, or Cary 100 Bio spectrophotometer.
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3836674/#R7
-- links to assay reagents (substrates) pages.
--- List cost and quantity of substrate reagents, supplier, and catalog #
Ligand(s) determined from virtual screening.
NADH
catalog #: N4505
supplier: Sigma-Aldrich
Cost: $67.30 for 100MG
Structure (PDB or Homology model)
-- PDB #: 3TSB
3USB - docked with IMP
Current Inhibitors: mycophenolic acid, tiazofurin, ribavirin , mizoribine, rapamycin
Expression Information (has it been expressed in bacterial cells): yas...
Purification Method: Ni-NTA (has 6X His Tag)
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
mhhhhhhssgvdlgtenlyfqsnamweskfvkegltfddvllvpaksdvlprevsvktvl seslqlniplisagmdtvteadmaiamarqgglgiihknmsieqqaeqvdkvkrsesgvi sdpffltpehqvydaehlmgkyrisgvpvvnnlderklvgiitnrdmrfiqdysikisdv mtkeqlitapvgttlseaekilqkykieklplvdnngvlqglitikdiekviefpnsakd kqgrllvgaavgvtadamtridalvkasvdaivldtahghsqgvidkvkevrakypslni iagnvataeatkalieaganvvkvgigpgsicttrvvagvgvpqltavydcatearkhgi pviadggikysgdmvkalaagahvvmlgsmfagvaespgeteiyqgrqfkvyrgmgsvga mekgskdryfqegnkklvpegiegrvpykgpladtvhqlvgglragmgycgaqdleflre naqfirmsgaglleshphhvqitkeapnysl
*length of your protein in Amino Acids: 511aa
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 55kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 26485
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
ATGTGGGAATCTAAATTCGTTAAAGAGGGCCTCACCTTTGACGACGTCCTGCTCGTCCCG
61 GCAAAAAGCGATGTACTCCCTCGTGAGGTTTCTGTTAAAACCGTTCTGTCCGAATCCCTC
121 CAGCTGAATATCCCGCTGATCTCTGCGGGTATGGATACGGTGACTGAAGCTGACATGGCG
181 ATTGCGATGGCTCGCCAAGGCGGCCTCGGCATTATTCACAAAAACATGTCTATCGAGCAG
241 CAAGCTGAACAGGTTGACAAGGTTAAACGCTCTGAATCTGGCGTCATCTCCGACCCGTTC
301 TTCCTGACCCCGGAGCATCAAGTGTACGACGCCGAACACCTGATGGGTAAATACCGCATC
361 TCTGGTGTCCCTGTCGTGAACAACCTCGACGAACGTAAGCTGGTAGGTATCATCACTAAC
421 CGTGACATGCGTTTTATTCAGGACTACTCTATCAAGATTAGCGACGTTATGACGAAGGAA
481 CAGCTGATTACTGCGCCTGTTGGTACTACCCTCTCCGAGGCAGAAAAAATCCTGCAAAAA
541 TACAAAATTGAGAAACTGCCGCTCGTTGATAATAACGGTGTTCTCCAGGGTCTCATCACG
601 ATTAAAGACATTGAAAAAGTTATCGAGTTCCCTAACTCTGCGAAGGACAAACAGGGTCGT
661 CTCCTGGTTGGCGCTGCAGTGGGTGTGACGGCTGACGCAATGACTCGTATCGACGCGCTG
721 GTTAAAGCGTCTGTTGATGCAATTGTTCTGGACACGGCCCACGGCCACTCCCAGGGCGTA
781 ATCGACAAAGTGAAGGAGGTTCGTGCGAAATACCCTTCTCTCAACATTATCGCGGGCAAC
841 GTAGCCACCGCTGAGGCTACTAAAGCGCTGATTGAGGCGGGTGCGAACGTTGTAAAAGTT
901 GGTATTGGTCCGGGTTCCATTTGCACGACTCGTGTAGTCGCAGGCGTTGGTGTGCCGCAA
961 CTGACTGCGGTTTACGACTGCGCGACCGAAGCCCGTAAACATGGCATCCCGGTAATCGCA
1021 GACGGCGGCATCAAATACTCTGGCGATATGGTAAAGGCACTGGCTGCTGGTGCACACGTA
1081 GTTATGCTCGGCTCTATGTTCGCCGGTGTTGCAGAGTCTCCGGGCGAAACTGAAATCTAC
1141 CAGGGCCGTCAATTCAAAGTATACCGTGGTATGGGTTCTGTGGGCGCTATGGAGAAAGGT
1201 TCCAAAGACCGTTACTTCCAGGAGGGTAACAAAAAACTCGTGCCGGAAGGTATTGAAGGT
1261 CGTGTACCGTACAAAGGTCCGCTCGCTGACACTGTACACCAGCTCGTCGGTGGTCTCCGT
1321 GCGGGCATGGGTTATTGTGGTGCGCAAGACCTCGAATTCCTCCGTGAGAATGCGCAGTTC
1381 ATCCGCATGAGCGGTGCTGGTCTGCTGGAATCTCACCCTCACCACGTGCAGATCACGAAA
1441 GAGGCTCCGAATTACAGCCTGTAA
*GC% Content for gene: 53.2%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
TACTTCCAATCCATGTGGGAATCTAAATTCGTTAAAGAGGGCCTCACCTTTGACGACGTCCTGCTCGTCCCG
GCAAAAAGCGATGTACTCCCTCGTGAGGTTTCTGTTAAAACCGTTCTGTCCGAATCCCTC
CAGCTGAATATCCCGCTGATCTCTGCGGGTATGGATACGGTGACTGAAGCTGACATGGCG
ATTGCGATGGCTCGCCAAGGCGGCCTCGGCATTATTCACAAAAACATGTCTATCGAGCAG
CAAGCTGAACAGGTTGACAAGGTTAAACGCTCTGAATCTGGCGTCATCTCCGACCCGTTC
TTCCTGACCCCGGAGCATCAAGTGTACGACGCCGAACACCTGATGGGTAAATACCGCATC
TCTGGTGTCCCTGTCGTGAACAACCTCGACGAACGTAAGCTGGTAGGTATCATCACTAAC
CGTGACATGCGTTTTATTCAGGACTACTCTATCAAGATTAGCGACGTTATGACGAAGGAA
CAGCTGATTACTGCGCCTGTTGGTACTACCCTCTCCGAGGCAGAAAAAATCCTGCAAAAA
TACAAAATTGAGAAACTGCCGCTCGTTGATAATAACGGTGTTCTCCAGGGTCTCATCACG
ATTAAAGACATTGAAAAAGTTATCGAGTTCCCTAACTCTGCGAAGGACAAACAGGGTCGT
CTCCTGGTTGGCGCTGCAGTGGGTGTGACGGCTGACGCAATGACTCGTATCGACGCGCTG
GTTAAAGCGTCTGTTGATGCAATTGTTCTGGACACGGCCCACGGCCACTCCCAGGGCGTA
ATCGACAAAGTGAAGGAGGTTCGTGCGAAATACCCTTCTCTCAACATTATCGCGGGCAAC
GTAGCCACCGCTGAGGCTACTAAAGCGCTGATTGAGGCGGGTGCGAACGTTGTAAAAGTT
GGTATTGGTCCGGGTTCCATTTGCACGACTCGTGTAGTCGCAGGCGTTGGTGTGCCGCAA
CTGACTGCGGTTTACGACTGCGCGACCGAAGCCCGTAAACATGGCATCCCGGTAATCGCA
GACGGCGGCATCAAATACTCTGGCGATATGGTAAAGGCACTGGCTGCTGGTGCACACGTA
GTTATGCTCGGCTCTATGTTCGCCGGTGTTGCAGAGTCTCCGGGCGAAACTGAAATCTAC
CAGGGCCGTCAATTCAAAGTATACCGTGGTATGGGTTCTGTGGGCGCTATGGAGAAAGGT
TCCAAAGACCGTTACTTCCAGGAGGGTAACAAAAAACTCGTGCCGGAAGGTATTGAAGGT
CGTGTACCGTACAAAGGTCCGCTCGCTGACACTGTACACCAGCTCGTCGGTGGTCTCCGT
GCGGGCATGGGTTATTGTGGTGCGCAAGACCTCGAATTCCTCCGTGAGAATGCGCAGTTC
ATCCGCATGAGCGGTGCTGGTCTGCTGGAATCTCACCCTCACCACGTGCAGATCACGAAA
GAGGCTCCGAATTACAGCCTGTAACAGTAAAGGTGGATA
*GC% Content for gene (codon optimized): 52.1%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
link to DNA Works output text file
Primer design results for 'tail' primers (this is just 2 sequences):